Receptor-regulated Ca2+ Entry Pathways in Smooth Muscle

平滑肌中受体调节的 Ca2 进入途径

• 批准号: 9037968
• 负责人: Mohamed Trebak
• 金额: $ 20.44万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-19 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9037968
• 关键词: Receptor; regulated; Ca2+; Entry; Pathways

DESCRIPTION (provided by applicant): Vascular smooth muscle cell (VSMC) proliferation and migration are essential for vascular development, angiogenesis and repair; their dysfunction contributes to vascular diseases such as atherosclerosis, hypertensive microvessel remodeling and Leiomyosarcomas. In these diseases VSMC switch from a quiescent differentiated and contractile phenotype to a synthetic proliferative and migratory phenotype, a condition that can be recapitulated in culture. There is clear evidence that this VSMC phenotypic modulation is of paramount clinical importance in atherosclerosis and other vascular occlusive diseases, yet the molecular mechanisms of this modulation remain incompletely understood. VSMC phenotypic modulation is accompanied by a change in ion channel expression: synthetic VSMC downregulate the expression of L-type Ca2+ channels and upregulate that of canonical transient receptor potential (TRPC) channels. Our preliminary studies have demonstrated an increase in TRPC6 and the newly discovered calcium sensor STIM1, and calcium channels Orai1 and Orai3 expression in synthetic cultured rat aortic VSMC, as compared to quiescent freshly isolated cells. We hypothesize that STIM1 is a master regulator of Ca2+ signaling in VSMC required for Orai1, Orai3 and TRPC6 channel function and that increased Ca2+ entry as a result of STIM1, Orai and TRPC upregulation contribute to VSMC proliferation and migration in disease. In support of this hypothesis we found that STIM1 knockdown using silencing RNA (siRNA) inhibited VSMC proliferation in culture and we revealed agonist-specific activation of distinct Ca2+ channels and remarkable STIM1 versatility in regulating these channels. Indeed, knockdown of STIM1 in synthetic VSMC abrogated the function of: i) PDGF-activated Orai1 channels; ii) thrombin-activated channels contributed by heteromultimeric Orai1/3 and iii) Diacylglycerol (DAG)-activated TRPC6 channels. In Aim1 we will biophysically characterize thrombin-activated Ca2+ entry pathways using whole cell patch clamp and determine the role of STIM1 oligomerization, cellular localization and interaction with Orai1 and Orai3 in the activation of this Ca2+ entry pathway. In Aim2 we will determine whether sarcoplasmic reticulum- or plasma membrane- associated STIM1 is involved in TRPC6 activation by DAG, examine the role of STIM1 oligomerization and the interaction of STIM1 and TRPC6 by FRET microscopy and the interaction of their native counterparts using co-immunoprecipitations. In Aim3 we will test the hypothesis that Orai1, Orai3 and TRPC6 upregulation also occurs in vivo in a rat model of vascular injury and determine the effect on neointimal formation of in vivo silencing of these proteins using adenovirus encoding siRNA. The results from this proposal will generate a better understanding of VSMC physiology and unveil novel targets for drug therapy aimed at controlling VSMC proliferation and migration that occur during vascular diseases such as atherosclerosis and hypertension.

描述（由申请方提供）：血管平滑肌细胞（VSMC）增殖和迁移对于血管发育、血管生成和修复至关重要;其功能障碍导致血管疾病，如动脉粥样硬化、高血压微血管重塑和平滑肌瘤。在这些疾病中，VSMC从静止的分化和收缩表型转变为合成的增殖和迁移表型，这种情况可以在培养中重现。有明确的证据表明，这种VSMC表型调制在动脉粥样硬化和其他血管闭塞性疾病中具有极其重要的临床意义，但这种调制的分子机制仍不完全清楚。 VSMC表型调节伴随着离子通道表达的变化：合成VSMC下调L型Ca 2+通道的表达，上调经典瞬时受体电位（TRPC）通道的表达。我们的初步研究表明，TRPC 6和新发现的钙传感器STIM 1，和钙通道Orai 1和Orai 3在合成培养的大鼠主动脉VSMC的表达增加，与静止的新鲜分离的细胞相比。我们假设STIM 1是VSMC中Orai 1、Orai 3和TRPC 6通道功能所需的Ca 2+信号传导的主要调节剂，并且STIM 1、奥赖和TRPC上调导致的Ca 2+内流增加有助于疾病中VSMC的增殖和迁移。为了支持这一假设，我们发现使用沉默RNA（siRNA）敲低STIM 1抑制培养中的VSMC增殖，并且我们揭示了不同Ca 2+通道的激动剂特异性激活和STIM 1在调节这些通道中的显著多功能性。事实上，在合成的VSMC中敲低STIM 1消除了以下通道的功能：i）PDGF激活的Orai 1通道; ii）由异源多聚体Orai 1/3贡献的凝血酶激活的通道和iii）二酰基甘油（DAG）激活的TRPC 6通道。在Aim 1中，我们将使用全细胞膜片钳技术对凝血酶激活的Ca 2+进入途径进行生物药理学表征，并确定STIM 1寡聚化、细胞定位以及与Orai 1和Orai 3相互作用在该Ca 2+进入途径激活中的作用。在Aim 2中，我们将确定肌浆网或质膜相关的STIM 1是否参与DAG激活TRPC 6，通过FRET显微镜检查STIM 1寡聚化的作用和STIM 1与TRPC 6的相互作用，以及使用免疫共沉淀的天然对应物的相互作用。在Aim 3中，我们将检验Orai 1、Orai 3和TRPC 6上调也在血管损伤的大鼠模型中体内发生的假设，并确定使用腺病毒编码siRNA体内沉默这些蛋白质对新生内膜形成的影响。该提案的结果将更好地了解VSMC生理学，并揭示药物治疗的新靶点，旨在控制动脉粥样硬化和高血压等血管疾病期间发生的VSMC增殖和迁移。